Srinivasan Damodaran

Formation of Whey Protein Isolate (WPI)−Dextran Conjugates in Aqueous Solutions

The conjugation reaction between whey protein isolate (WPI) and dextran in aqueous solutions via the initial stage of the Maillard reaction was studied. The covalent attachment of dextran to WPI was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with both protein and carbohydrate staining. The formation of WPI-dextran conjugates was monitored by a maximum absorbance peak at approximately 304 nm using difference UV spectroscopy. The impact of various processing conditions on the formation of WPI-dextran conjugates was investigated. The conjugation reaction was promoted by raising the temperature from 40 to 60 degrees C, the WPI concentration from 2.5 to 10%, and the dextran concentration from 10 to 30% and lowering the pH from 8.5 to 6.5. The optimal conjugation conditions chosen from the experiments were 10% WPI-30% dextran and pH 6.5 at 60 degrees C for 24 h. WPI-dextran conjugates were stable under the conditions studied.

Kinetics of adsorption of proteins at interfaces: role of protein conformation in diffusional adsorption

To elucidate the role of protein conformation in the kinetics of adsorption at interfaces, seven structural intermediates of bovine serum albumin were prepared and their adsorption at the air/water interface was studied. Molecular area calculations indicated two distinct molecular processes, the first being the creation of an area, delta A1, for anchoring the molecule during the initial phase of adsorption and the second being the delta A2 cleared during subsequent reorientation and rearrangement of adsorbed molecules at the interface. The delta A1 values for all the albumin intermediates were the same, indicating that the initial work pi delta A1 needed to anchor the molecule at the interface was independent of solution conformation of the protein. Unlike delta A1, delta A2 exhibited a bell-shaped relationship with the extent of refolded state of the intermediates. Calculation of diffusion coefficients indicated that greater the unfolded state of the albumin intermediate, the greater was the diffusion coefficient. It is shown that the simple diffusion theory is inadequate to explain quantitatively the kinetics of protein adsorption. Specific, conformation-dependent, solute-solvent and solute-interface interactions also seem to influence the kinetics of adsorption of proteins.

Physicochemical and Emulsifying Properties of Whey Protein Isolate (WPI)−Dextran Conjugates Produced in Aqueous Solution

The physicochemical and emulsifying properties of protein and polysaccharide conjugates prepared under mild conditions were investigated. The covalently linked conjugates of whey protein isolate (WPI) and dextran (DX, 440 kDa) were produced by incubating aqueous solutions containing 10% WPI and 30% DX at pH 6.5 and 60 degrees C for 48 h. After purification by anion-exchange chromatography and affinity chromatography, the conjugate had a weight-average molecular weight (M(w)) of 531 kDa and a radius of gyration (R(g)) of 30 nm as determined by size exclusion chromatography-multiangle laser light scattering (SEC-MALLS); the molar binding ratio of WPI to DX was calculated to be approximately 1:1. The purified conjugate had significantly improved heat stability when subjected to 80 degrees C for 30 min and remained soluble over a range of pH from 3.2 to 7.5 and ionic strengths from 0.05 to 0.2 M in contrast to native WPI. The emulsifying ability and emulsion stability made with WPI-DX conjugate were also improved compared to WPI and gum arabic (an emulsifier containing naturally derived glycoproteins).

Interfaces, Protein Films, and Foams

Publisher Summary This chapter presents an overview of the thermodynamics of surfaces with specific reference to the air–water interface and discusses the molecular aspects of the kinetics and thermodynamics of proteins at the air–water interface. A discussion of the properties and the factors affecting the stability of protein-stabilized foams is presented in the chapter. Although all proteins are amphipathic, proteins differ very much in their surface activity. The differences in the surface-active properties of various proteins cannot simply be attributed to nonspecific variations in the amphipathicity of proteins, because, within reasonable limits, most proteins exhibit similar distribution of hydrophobic and hydrophilic residues. All of these molecular properties collectively influence the film-forming and foaming properties of proteins. The elucidation of the role of each of these molecular properties on adsorption and film formation of proteins at interfaces is the cherished dream of physical chemists. Such an understanding has been difficult to attain not because of conceptual difficulties but because of the interdependency of each of the molecular properties of the protein on others.

Estimation of disulfide bonds using 2-nitro-5-thiosulfobenzoic acid: limitations

Recently an elegant method for the quantification of the number of disulfide bonds in proteins and peptides has been reported [T.W. Thannhauser, Y. Konishi, and H.A. Scheraga (1984) Anal. Biochem. 138, 181-188]. The method is based on the quantification of 2-nitro-5-thiobenzoate (NTB) formed from the reaction of 2-nitro-5-thiosulfobenzoate with disulfides in the presence of excess sodium sulfite. Here it is reported that the NTB anion undergoes photochemical reaction with excess sulfite in the system, which results in the rapid disappearance of absorbance at 412 nm in the presence of light. The nonchromophoric derivative of this photochemical reaction is tentatively identified as a sulfo derivative of NTB. Based on these observations it is suggested that, for the quantification of disulfide bonds using the NTSB method, the assay should be carried out in the dark.

pH-stability and thermal properties of microbial transglutaminase-treated whey protein isolate.

Whey protein isolate (WPI) was treated to various extents using microbial transglutaminase (MTGase) and changes in pH-stability and thermal stability of its protein components were investigated. The MTGase treatment significantly increased the denaturation temperature (T(d)) of beta-lactoglobulin in WPI, from 71.84 degrees C in the untreated sample to 78.50 degrees C after 30 h of incubation with MTGase. The enthalpy change of denaturation of WPI did not change upon cross-linking, indicating that the increase in T(d) was primarily due to covalent cross-linking and not due to an increase in nonpolar interactions within the protein. The surface hydrophobicity (S(o)) of the protein decreased upon cross-linking; however, this decrease was not due to burial of the surface hydrophobic cavities in the protein interior, but due to occlusion of the hydrophobic cavities to the fluorescent probes. Fluorescence emission and circular dichroism spectroscopic analyses revealed no major changes in the secondary and tertiary conformations as a result of cross-linking. However, unlike native WPI, the cross-linked protein exhibited a U-shaped pH-stability profile with maximum turbidity at pH 4.0-4.5. The study revealed that even though enzymatic cross-linking significantly improved the T(d) of beta-lactoglobulin in WPI without causing major structural changes, a reduction in the hydrophilic-hydrophobic balance of the protein surface as a result of elimination of the positive charge on lysyl residues caused precipitation at pH 4.0-4.5.

Heat-induced conformational changes in phaseolin and its relation to proteolysis

Heat-induced conformational changes and their relationship to trypsinolysis of phaseolin, the major storage globulin of Phaseolus vulgaris , were studied using far- and near-UV circular dichroism (CD), second-derivative UV absorption spectroscopy and intrinsic fluorescence quenching of Trp fluorescence by an ionic (iodide) and neutral (acrylamide) quencher. Thermal denaturation did not cause any significant changes in the secondary structure of phaseolin. However, near-UV CD data indicated a 0.8–3-fold decrease in the intensity of Phe and Tyr peaks. At least 22 out of 31 Tyr residues in the native phaseolin, while 29 in the 30-min heated phaseolin were exposed to aqueous solvent. The fluorescent Trp residues in both the native and heated phaseolins were little accessible to iodide, while upon heating, 70–90% of the fluorescence was quenched by acrylamide. The surface hydrophobicity of the protein increased 7–9-fold upon thermal denaturation. The results presented here suggest that a disruption upon heating of the tertiary and quaternary structures of phaseolin, and not changes in the secondary structure, is crucial for its enhanced susceptibility to trypsin.

Metal-chelating properties and biodegradability of an ethylenediaminetetraacetic acid dianhydride modified soy protein hydrogel

The heavy-metal chelating properties of a soy protein based hydrogel, prepared by crosslinking an ethylenediaminetetraacetic acid dianhydride (EDTAD) modified soy protein isolate (SPI), have been studied. The equilibrium binding capacities of the divalent calcium, zinc, mercury, and lead ions by the gel were 0.70, 0.65, 0.95, and 0.70 mmol per gram of dry gel, respectively. The distribution ratio of metal ions between the gel and the solution was in the range of 370 to 15,000 mL/g, depending on the initial metal concentration. A positive relationship between the carboxyl group content of EDTAD-modified SPI and the metal-binding ability of the gel was observed; the optimum metal binding occurred at 25°C. The metal-binding ability increased with increasing pH, in the range where the solubility of the metal ions was not affected by the pH. In binary metal ion solutions, the metal ions adsorbed to the gel in a competitive fashion, influenced by the initial ion concentration. The EDTAD-modified protein hydrogel was readily degraded by proteolytic enzymes and was biodegraded in a fungal overgrowth test. The EDTAD-SPI hydrogel was completely degraded after a 28-day incubation with fungal spores.

Equilibrium swelling properties of a novel ethylenediaminetetraacetic dianhydride (EDTAD)-modified soy protein hydrogel

A novel pH and ionic strength-sensitive protein-based hydrogel was synthesized via cross-linking ethylenediaminetetraacetic dianhydride-modified soy protein isolate (EDTAD–SPI) with glutaraldehyde. Incorporation of ionizable carboxyl groups into soy proteins increased the net negative charge of the protein and caused extensive unfolding of the protein structure. The EDTAD–SPI hydrogel was capable of imbibing 80-300 g water per g dry gel after centrifuging at 214g, depending on the extent of modification, protein structure, crosslinking density, protein concentration during the crosslinking step, gel particle size, and environ-mental conditions, such as temperature, pH, and ionic strength. The protein concentration used during the crosslinking step was found to be the most important factor affecting the water uptake of the gel. The lower the protein concentration, the higher was the water uptake at 214g. The hydrogel was highly sensitive to pH and exhibited reversible swelling when sequentially exposed to water and 0.15M NaCl.

Influence of protein conformation on its adaptability under chaotropic conditions

The thermostability of serum albumin and beta-lactoglobulin in various salt solutions was studied using differential scanning calorimetry. Below 1.0 M salt concentrations, the relative effectiveness of various sodium salts on increasing the thermostability of beta-lactoglobulin followed the classic Hofmeister or lyotropic series, i.e. SO2-(4) greater than Cl- greater than Br- greater than ClO-4 greater than SCN-; however, in the case of serum albumin the above order was reversed, i.e. ClO-4 greater than SCN- greater than Br- greater than Cl- greater than SO2-(4), indicating that the thermostability of serum albumin was higher in chaotropic solution conditions. Circular dichroic analysis of serum albumin in NaClO4 solutions revealed that the alpha-helical content of the protein increased from 59% to 73% in 1.0 M NaClO4; no similar increase in secondary structure was observed for beta-lactoglobulin. These observations contradicted the general notion that the chaotropic effect of neutral salts on the stability of macromolecules is independent of any details of the macromolecular conformation itself. The results presented here indicate that the predisposition of the native conformation of a protein per se might affect whether the protein would undergo stabilization or destabilization (i.e. conformational adaptability) under moderate chaotropic solution conditions.